Accurate determination of the presence, identity, concentration, and/or quantity of an ionized species in a sample is critically important in many fields. Most techniques used in such analyses involve ionization of species in a fluid sample prior to introduction into the analytical equipment employed. The choice of ionization method will depend on the nature of the sample and the analytical technique used, and many ionization methods are available, including, without limitation, chemical ionization, electron impact ionization, desorption chemical ionization, and atmospheric pressure ionization, including electrospray ionization and atmospheric pressure chemical ionization.
The presence of contaminants in biological samples undergoing analysis is obviously problematic for a number of reasons. Contaminants might cause interference with the analytical procedure, chemically or physically altering the sample or the analyte itself. Contaminants may also be mistaken for analyte or vice versa, such that the concentration measured concentration of analyte may be significantly higher or lower than the actual concentration. The same problem can be caused by necessary components of the fluid sample, such as buffer salts, surfactants, and other species that may be essential for biochemical steps preceding the analysis.
Mass spectrometry is a well-established technique that involves the detection of an analyte in ionized form. In this technique, sample molecules are ionized and the resulting ions are sorted by mass-to-charge ratio. For analytes contained in a fluid sample, the sample is typically converted to an aerosol that undergoes desolvation, vaporization, and ionization in order to form fluid ions.
The presence of non-analyte species can be particularly problematic in mass spectrometry, where analyte concentration may be very low and the number and concentration of contaminants and interfering components may be relatively high. One study has documented over 650 contaminants and interfering components frequently found in biological samples undergoing mass spectrometric analysis. B. O. Keller et al. (2008), “Interferences and contaminants encountered in modern mass spectrometry,” Anal. Chim. Acta 627(1):71-81. These include salts, buffering agents, endogenous compounds, surfactants, drugs, metabolites, and proteins. When these species reduce detection sensitivity by decreasing the signal-to-noise ratio and give rise to a flawed analysis, the problem has been characterized as “ion suppression.” See Weaver et al. (2006) Rapid Communications in Mass Spectrometry 20:2559-64.
It has been postulated that “the main cause of ion suppression is a change in the spray droplet solution properties caused by the presence of nonvolatile or less volatile solutes,” i.e., solutes that are nonvolatile or less volatile than the analyte; see Annesley (2003) “Ion Suppression in Mass Spectrometry,” in Clinical Chemistry 49(7):1041-1044, citing King et al. (2000) J. Am. Soc. Mass Spectrom. 11:942-50. The reference explains that the nonvolatile or less volatile contaminants and components change the efficiency of droplet formation or droplet volatilization, which in turn affects the quantity of charged analyte in the gas phase that ultimately reaches the detector. Annesley cites studies showing that molecules of higher mass tend to suppress the signal of smaller molecules and that more polar analytes are susceptible to ion suppression. Annesley at 1042, citing Sterner et al. (2000) J. Mass Spectrom. 35:385-91 and Bonfiglio et al. (1999) Rapid Commun. Mass Spectrom. 13:1175-85. Weaver et al. cites several possible mechanisms underlying ion suppression: (1) competition for charge between analyte and ion-suppressing agent, leading to reduced ionization of analyte; (2) large concentrations of ion-suppressing agents causing an increase in surface tension as well as an increase in droplet viscosity, in turn resulting in decreased evaporation efficiency; and (3) gas phase reactions between the ionized analyte and other species in the sample, resulting in an overall loss of charge from the analyte ions. Weaver et al. at 2562.
As electrospray ionization (ESI) has a relatively complex ionization mechanism, relying heavily on droplet charge excess, there are additional factors to consider when exploring the cause of ion suppression and potential solutions. It has been widely observed that for many analytes, at high concentrations, ESI exhibits a loss of detector response linearity, perhaps due to reduced charge excess caused by analyte saturation at the droplet surface, inhibiting subsequent ejection of gas phase ions from further inside the droplet. Thus, competition for space and/or charge may be considered as a source of ion suppression in ESI. Both physical and chemical properties of analytes (e.g. basicity and surface activity) determine their inherent ionization efficiency. Biological sample matrices naturally tend to contain many endogenous species with high basicity and surface activity, and the total concentration of these species in the sample will thus quickly reach levels at which ion suppression can be expected.
Another explanation of ion suppression in ESI considers the physical properties of the droplet itself rather than the species present. As noted above, high concentrations of interfering components give rise to increased surface tension and viscosity that in turn reduce evaporation efficiency, and this is known to have a marked effect on ionization efficiency.
An additional theory to explain ion suppression in ESI relates to the presence of non-volatile species that can either cause co-precipitation of analyte in the droplet (thus preventing ionization) or prevent the contraction of droplet size to the critical radius required for ion evaporation and/or charge residue mechanisms to form gas phase ions efficiently. It should also be pointed out that the degree of ion suppression may be dependent on the concentration of the analyte being monitored, and with the ever-increasing demand to lower detection threshold, ion suppression may become a more and more serious problem.
Ion suppression has primarily been addressed by de-salting the fluid sample using dialysis, liquid chromatography, solid-phase extraction, or ion exchange. These processes require time, materials, and equipment, and can reduce the available quantity of an already small sample. In addition, certain ionic or ionizable species may be essential to maintain in the sample, such as buffer systems.
An ideal method to address ion suppression would:
Eliminate the need for additional process steps and materials, including clean-up and de-salting;
Eliminate the need for additional processing time;
Be adaptable to use with very small sample sizes, consume a small portion of the small sample size, allow for detection of low analyte concentrations, and be composed of very small droplets; and
Be capable of implementation in a high speed analytical system such as high throughput mass spectrometry, optimally enabling analysis of up to at least 50,000 samples per day or more; and
Eliminate the need for pre-analysis “clean-up” of the sample to remove contaminants and interfering components.